Broad spectrum thiopeptide recognition specificity of the Streptomyces lividans TipAL protein and its role in regulating gene expression

Microbial metabolites isolated in screening programs for their ability to a ctivate transcription of the tipA promoter (ptipA) in Streptomyces lividans define a class of cyclic thiopeptide antibiotics having dehydroalanine sid e chains ("tails"). Here we show that such compounds of heterogeneous prima ry structure (representatives tested: thiostrepton, nosiheptide, berninamyc in, promothiocin) are all recognized by TipAS and TipAL, two in-frame trans lation products of the tipA gene. The N-terminal helix-turn-helix DNA bindi ng motif of TipAL is homologous to the MerR family of transcriptional activ ators, while the C terminus forms a novel ligand-binding domain. ptipA indu cers formed irreversible complexes in vitro and in vivo (presumably covalen t) with TipAS by reacting with the second of the two C-terminal cysteine re sidues. Promothiocin and thiostrepton derivatives in which the dehydroalani ne side chains were removed lost the ability to modify TipAS. They were abl e to induce expression of ptipA as well as the tipA gene, although with red uced activity. Thus, TipA required the thiopeptide ring structure for recog nition, while the tail served either as a dispensable part of the recogniti on domain and/or locked thiopeptides onto TipA proteins, thus leading to an irreversible transcriptional activation. Construction and analysis of a di sruption mutant showed that tipA was autogenously regulated and conferred t hiopeptide resistance. Thiostrepton induced the synthesis of other proteins , some of which did not require tipA.